Glutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mammalian brain. Changes in synaptic efficacy underlie aspects of learning and memory and help sculpt neural networks in development. Furthermore, GluRs have been implicated in the pathophysiology of many diseases including schizophrenia, stroke, epilepsy and neurodegenerative diseases such as Alzheimer disease. Understanding synaptic regulation of GluR will therefore provide valuable insight into the mechanisms for brain physiology and disease. The major ionotropic GluRs include the -amino-3hydroxyl-5methyl4-isoxazolepropionate receptors (AMPARs) and NmethylDaspartate receptors (NMDARs). AMPARs are loosely associated with the synapse, and their density at synapses is tightly controlled by neuronal activity. Although many studies have focused on the interactions of AMPARs with molecules involved in vesicle fusion, not much is known about the role of AMPARbinding proteins in synaptic plasticity. The protein stargazin is a GluRbinding protein that is mutated in stargazer, a strain of mice with epilepsy and ataxia. The primary deficit in this mouse is abnormal targeting of GluR to synapses. The goal of this research plan is to explore the role of the protein stargazin in the regulation of AMPAR synaptic targeting. The primary hypothesis of the proposed research is that stargazin mediates synaptic targeting of AMPARs, and phosphorylation of stargazin modulates AMPAR localization and function in synaptic plasticity. The Specific Aims of the proposed project are: 1) To characterize the interaction between stargazin and GluR subunits, 2) to analyze the role of stargazin phosphorylation in AMPAR synaptic targeting, and 3) to identify binding partners of stargazin and phosphorylated stargazin. Experimental design and methods to accomplish the first Aim include standard molecular biology techniques to generate deletion constructs of stargazin and GluR and coinmunoprecipitation from transfected cells. The second Aim will involve in vitro phosphorylation of peptide substrates, immunohistochemistry in transfected heterologous cells and neurons, and in vitro hippocampal slice preparation to evaluate the role of stargazin phosphorylation in longterm potentiation (LTP). The third Aim will involve the yeast 2hybrid screening method, followed by cloning of binding partners and analysis by immunoprecipitation and immunohistochemistry of brain slices and cortical neurons. The experiments described in the research plan should allow a detailed account of the basic mechanism of GluR targeting to synapses, and posssibly provide insight into mechanisms by which GluR targeting is deranged in neurological disease.